Physical isolation of nascent RNA chains transcribed by RNA polymerase II: evidence for cotranscriptional splicing

In order to examine whether splicing can occur cotranscriptionally in mammalian nuclei, we mapped exon-intron boundaries on nascent RNA chains transcribed by RNA polymerase II. A procedure that allows fractionation of nuclei into a chromatin pellet containing DNA, histones, and ternary transcription complexes and a supernatant containing the bulk of the nonhistone proteins and RNAs that are released from their DNA templates was developed. The transcripts of the genes encoding DBP, a transcriptional activator protein, and HMG coenzyme A reductase recovered from the chromatin pellet and the supernatant were analyzed by S1 nuclease mapping. The large majority of the RNA molecules from the pellet appeared to be nascent transcripts, since, in contrast to the transcripts present in the supernatant, they were not cleaved at the polyadenylation site but rather contained heterogeneous 3' termini encompassing this site. Splicing intermediates could be detected among nascent and released transcripts, suggesting that splicing occurs both cotranscriptionally and posttranscriptionally. Our results also indicate that polyadenylation is not required for the splicing of the last DBP intron. In addition to allowing detailed structural analysis of nascent RNA chains, the physical isolation of nascent transcripts also yields reliable measurements of relative transcription rates.

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Active RNA polymerases are localized within discrete transcription “factories' in human nuclei

Journal of Cell Science ◽

10.1242/jcs.109.6.1427 ◽

1996 ◽

Vol 109 (6) ◽

pp. 1427-1436 ◽

Cited By ~ 9

Author(s):

F.J. Iborra ◽

A. Pombo ◽

D.A. Jackson ◽

P.R. Cook

Keyword(s):

Electron Microscopy ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Hela Cells ◽

Rna Polymerases ◽

Transcription Factories ◽

Nascent Rna ◽

Nascent Transcripts ◽

Typical Site

Nascent transcripts in permeabilized HeLa cells were elongated by approximately 30–2,000 nucleotides in Br-UTP or biotin-14-CTP, before incorporation sites were immunolabelled either pre- or post-embedding, and visualized by light or electron microscopy. Analogues were concentrated in approximately 2,100 (range 2,000-2,700) discrete sites attached to a nucleoskeleton and surrounded by chromatin. A typical site contained a cluster (diameter 71 nm) of at least 4, and probably about 20, engaged polymerases, plus associated transcripts that partially overlapped a zone of RNA polymerase II, ribonucleoproteins, and proteins rich in thiols and acidic groups. As each site probably contains many transcription units, these results suggest that active polymerases are confined to these sites, which we call transcription ‘factories’. Results are consistent with transcription occurring as templates slide past attached polymerases, as nascent RNA is extruded into the factories.

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Splicing-independent recruitment of spliceosomal small nuclear RNPs to nascent RNA polymerase II transcripts

The Journal of Cell Biology ◽

10.1083/jcb.200706134 ◽

2007 ◽

Vol 178 (6) ◽

pp. 937-949 ◽

Cited By ~ 12

Author(s):

Snehal Bhikhu Patel ◽

Natalya Novikova ◽

Michel Bellini

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Messenger Rnas ◽

Stem Loop ◽

Small Nuclear Ribonucleoprotein ◽

Small Nuclear Rnas ◽

Transcriptional Units ◽

Necessary And Sufficient ◽

Nuclear Ribonucleoprotein ◽

Nascent Transcripts

In amphibian oocytes, most lateral loops of the lampbrush chromosomes correspond to active transcriptional sites for RNA polymerase II. We show that newly assembled small nuclear ribonucleoprotein (RNP [snRNP]) particles, which are formed upon cytoplasmic injection of fluorescently labeled spliceosomal small nuclear RNAs (snRNAs), target the nascent transcripts of the chromosomal loops. With this new targeting assay, we demonstrate that nonfunctional forms of U1 and U2 snRNAs still associate with the active transcriptional units. In particular, we find that their association with nascent RNP fibrils is independent of their base pairing with pre–messenger RNAs. Additionally, stem loop I of the U1 snRNA is identified as a discrete domain that is both necessary and sufficient for association with nascent transcripts. Finally, in oocytes deficient in splicing, the recruitment of U1, U4, and U5 snRNPs to transcriptional units is not affected. Collectively, these data indicate that the recruitment of snRNPs to nascent transcripts and the assembly of the spliceosome are uncoupled events.

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DNA polymerase ε associates with the elongating form of RNA polymerase II and nascent transcripts

FEBS Journal ◽

10.1111/j.1742-4658.2006.05544.x ◽

2006 ◽

Vol 273 (24) ◽

pp. 5535-5549 ◽

Cited By ~ 6

Author(s):

Anna K. Rytkönen ◽

Tomi Hillukkala ◽

Markku Vaara ◽

Miiko Sokka ◽

Maarit Jokela ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Dna Polymerase ◽

Nascent Transcripts

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CDK9 and PP2A regulate the link between RNA polymerase II transcription termination and RNA maturation.

10.1101/2021.06.21.449289 ◽

2021 ◽

Author(s):

Michael Tellier ◽

Justyna Zaborowska ◽

Jonathan Neve ◽

Takayuki Nojima ◽

Svenja Hester ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Premature Termination ◽

Transcription Termination ◽

Elongation Factor ◽

Protein Coding ◽

Pol Ii ◽

Working Together ◽

Rna Maturation ◽

Nascent Transcripts

CDK9 is a critical kinase required for the productive transcription of protein-coding genes by RNA polymerase II (pol II) in higher eukaryotes. Phosphorylation of targets including the elongation factor SPT5 and the carboxyl-terminal domain (CTD) of RNA pol II allows the polymerase to pass an early elongation checkpoint (EEC), which is encountered soon after initiation. In addition to halting RNA polymerase II at the EEC, CDK9 inhibition also causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, suggesting that CDK9 and PP2A, working together, regulate the coupling of elongation and transcription termination to RNA maturation. Our phosphoproteomic analyses, using either DRB or an ATP analog-sensitive CDK9 cell line confirm the splicing factor SF3B1 as an additional key target of this kinase. CDK9 inhibition causes loss of interaction of splicing and export factors with SF3B1, suggesting that CDK9 also helps to co-ordinates coupling of splicing and export to transcription.

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TASOR epigenetic repressor cooperates with a CNOT1 RNA degradation pathway to repress HIV

10.1101/2020.11.22.386656 ◽

2020 ◽

Author(s):

Roy Matkovic ◽

Marina Morel ◽

Pauline Larrous ◽

Benjamin Martin ◽

Fabienne Bejjani ◽

...

Keyword(s):

Gene Expression ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Rna Degradation ◽

Degradation Pathway ◽

Transcriptional Level ◽

Heterochromatin Formation ◽

Nascent Transcripts ◽

Ribosomal Dnas

AbstractThe Human Silencing Hub (HUSH) complex constituted of TASOR, MPP8 and Periphilin is involved in the spreading of H3K9me3 repressive marks across genes and transgenes such as ZNF encoding genes, ribosomal DNAs, LINE-1, Retrotransposons and Retroelements or the integrated HIV provirus1–5. The deposit of these repressive marks leads to heterochromatin formation and inhibits gene expression. The precise mechanisms of silencing mediated by HUSH is still poorly understood. Here, we show that TASOR depletion increases the accumulation of transcripts derived from the HIV-1 LTR promoter at a post-transcriptional level. By counteracting HUSH, Vpx from HIV-2 mimics TASOR depletion. With the use of a Yeast-Two-Hybrid screen, we identified new TASOR partners involved in RNA metabolism including the RNA deadenylase CCR4-NOT complex scaffold CNOT1. TASOR and CNOT1 interact in vivo and synergistically repress HIV expression from its LTR. In fission yeast, the RNA-induced transcriptional silencing (RITS) complex presents structural homology with HUSH. During transcription elongation by RNA polymerase II, RITS recruits a TRAMP-like RNA degradation complex composed of CNOT1 partners, MTR4 and the exosome, to ultimately repress gene expression via H3K9me3 deposit. Similarly, we show that TASOR interacts and cooperates with MTR4 and the exosome, in addition to CNOT1. We also highlight an interaction between TASOR and RNA Polymerase II, predominantly under its elongating state, and between TASOR and some HUSH-targeted nascent transcripts. Furthermore, we show that TASOR overexpression facilitates the association of the aforementioned RNA degradation proteins with RNA polymerase II. Altogether, we propose that HUSH operates at the transcriptional and post-transcriptional levels to repress HIV proviral gene expression.

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Specificity of RNA maturation pathways: RNAs transcribed by RNA polymerase III are not substrates for splicing or polyadenylation

Molecular and Cellular Biology ◽

10.1128/mcb.7.10.3602-3612.1987 ◽

1987 ◽

Vol 7 (10) ◽

pp. 3602-3612

Author(s):

S S Sisodia ◽

B Sollner-Webb ◽

D W Cleveland

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Rna Polymerase Iii ◽

Rnase H ◽

Intact Cells ◽

S1 Nuclease ◽

Polymerase Iii ◽

Hybrid Genes ◽

Rna Maturation

To analyze the specificity of RNA processing reactions, we constructed hybrid genes containing RNA polymerase III promoters fused to sequences that are normally transcribed by polymerase II and assessed their transcripts following transfection into human 293 cells. Transcripts derived from these chimeric constructs were analyzed by using a combined RNase H and S1 nuclease assay to test whether RNAs containing consensus 5' and 3' splicing signals could be efficiently spliced in intact cells, even though they were transcribed by RNA polymerase III. We found that polymerase III-derived RNAs are not substrates for splicing. Similarly, we were not able to detect poly(A)+ RNAs derived from genes that contained a polymerase III promoter linked to sequences that were necessary and sufficient to direct 3'-end cleavage and polyadenylation when transcribed by RNA polymerase II. Our findings are consistent with the view that in vivo splicing and polyadenylation pathways are obligatorily coupled to transcription by RNA polymerase II.

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